Extracellular vesicles in malaria: Pathogenesis, diagnosis and therapy.

Malaria is a life-threatening disease caused by parasites from the genus Plasmodium. Five species can cause malaria in humans, with Plasmodium vivax being the most common in many countries and Plasmodium falciparum having the highest lethality, which can lead to cerebral malaria. Extracellular vesicles (EVs) are in focus in malaria research to better understand pathogenesis, diagnosis, therapy, and prognosis. Malaria-causing parasites use EVs to transfer their molecules to host cells, a mechanism that significantly contributes to parasite survival and successful infection. EVs have thus emerged as an essential component of the immunopathological cascade of malaria, playing a pivotal role in disease progression and severity. This chapter discusses the epidemiology and pathogenesis of malaria and the role of EVs as new diagnostic and therapeutic tools, emphasizing their potential clinical significance.

Host 5-HT affects Plasmodium transmission in mosquitoes via modulating mosquito mitochondrial homeostasis.

Malaria parasites hijack the metabolism of their mammalian host during the blood-stage cycle. Anopheles mosquitoes depend on mammalian blood to lay eggs and to transmit malaria parasites. However, it remains understudied whether changes in host metabolism affect parasite transmission in mosquitoes. In this study, we discovered that Plasmodium infection significantly decreased the levels of the tryptophan metabolite, 5-hydroxytryptamine (5-HT), in both humans and mice. The reduction led to the decrease of 5-HT in mosquitoes. Oral supplementation of 5-HT to Anopheles stephensi enhanced its resistance to Plasmodium berghei infection by promoting the generation of mitochondrial reactive oxygen species. This effect was due to the accumulation of dysfunctional mitochondria caused by 5-HT-mediated inhibition of mitophagy. Elevating 5-HT levels in mouse serum significantly suppressed parasite infection in mosquitoes. In summary, our data highlight the critical role of metabolites in animal blood in determining the capacity of mosquitoes to control parasite infection.

Persistent bone marrow hemozoin accumulation confers a survival advantage against bacterial infection via cell-intrinsic Myd88 signaling.

Malaria remains a global health challenge, affecting millions annually. Hemozoin (Hz) deposition in the bone marrow disrupts hematopoiesis and modulates immune responses, but the mechanisms are not fully understood. Here, we show that persistent hemozoin deposition induces a sustained bias toward myelopoiesis, increasing peripheral myeloid cell numbers. Hz drives this process through a cell-intrinsic, MyD88-dependent pathway, enhancing chromatin accessibility of transcription factors such as Runx1 and Etv6 in granulocyte-macrophage progenitors. These findings are confirmed by intraosseous Hz injections and bone marrow chimeras. Single-cell RNA sequencing reveals increased reactive oxygen species production in monocytes from malaria-recovered mice, correlating with enhanced bactericidal capacity. This highlights an alternative aspect of post-malarial immunity and extends our understanding of trained immunity, suggesting that pathogen by-products like Hz can induce innate immune memory. These results offer insights into therapeutic strategies that harness trained immunity to combat infectious diseases.

NDV targets the invasion pathway in malaria parasite through cell surface sialic acid interaction.

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs.

A systematic review and meta-analysis of cortisol levels in Plasmodium infections.

Malaria has complex interactions with host physiology, including alterations in cortisol levels. Cortisol, a key hormone in the stress response, is known to be dysregulated in various infectious diseases. This systematic review and meta-analysis aimed to elucidate the relationship between Plasmodium infection and cortisol levels, shedding light on the intricate interplay between the parasite and the host's endocrine system. The methodological protocol for assessing cortisol levels in malaria patients was registered in PROSPERO (CRD42024496578), a widely recognized international prospective register of systematic reviews. This registration ensures transparency and minimizes the risk of bias in our research. A comprehensive search strategy was employed across major databases, including Embase, PubMed, Scopus, and Medline, to include studies that reported cortisol levels in infected patients. The qualitative synthesis was undertaken to synthesize the difference in cortisol levels between malaria-infected and uninfected individuals. The meta-analysis employed the random effects model in the quantitative synthesis to calculate the effect estimate. The review included a total of 20 studies, with a substantial number conducted in Africa, followed by Asia and South America. Most included studies (13/20, 65%) reported higher cortisol levels in infected patients than in uninfected patients. The meta-analysis confirmed significantly higher cortisol levels in infected patients compared to uninfected individuals (P < 0.0001, standardized mean difference (SMD): 1.354, 95% confidence interval: 0.913 to 1.795, I2: 88.3%, across 15 studies). Notably, the method for cortisol measurement and the type of blood sample used (serum or plasma) were significant moderators in the analysis, indicating that these factors may influence the observed relationship between Plasmodium infection and cortisol levels. The systematic review and meta-analysis confirmed that Plasmodium infection is associated with increased cortisol levels, highlighting the intricate relationship between the disease and the host stress response. These findings underscore the potential of cortisol as a supplementary biomarker for understanding the pathophysiological impact of malaria. By providing insights into the stress-related mechanisms of malaria, this comprehensive understanding can inform future research and potentially enhance disease management and treatment strategies, particularly in regions heavily burdened by malaria.

Endocytosis in malaria parasites: An ultrastructural perspective of membrane interplay in a unique infection model.

Malaria remains a major global threat, representing a severe public health problem worldwide. Annually, it is responsible for a high rate of morbidity and mortality in many tropical developing countries where the disease is endemic. The causative agent of malaria, Plasmodium spp., exhibits a complex life cycle, alternating between an invertebrate vector, which transmits the disease, and the vertebrate host. The disease pathology observed in the vertebrate host is attributed to the asexual development of Plasmodium spp. inside the erythrocyte. Once inside the red blood cell, malaria parasites cause extensive changes in the host cell, increasing membrane rigidity and altering its normal discoid shape. Additionally, during their intraerythrocytic development, malaria parasites incorporate and degrade up to 70 % of host cell hemoglobin. This mechanism is essential for parasite development and represents an important drug target. Blocking the steps related to hemoglobin endocytosis or degradation impairs parasite development and can lead to its death. The ultrastructural analysis of hemoglobin endocytosis on Plasmodium spp. has been broadly explored along the years. However, it is only recently that the proteins involved in this process have started to emerge. Here, we will review the most important features related to hemoglobin endocytosis and catabolism on malaria parasites. A special focus will be given to the recent analysis obtained through 3D visualization approaches and to the molecules involved in these mechanisms.

Melatonin as a Circadian Marker for Plasmodium Rhythms.

Plasmodium, a digenetic parasite, requires a host and a vector for its life cycle completion. Most Plasmodium species display circadian rhythmicity during their intraerythrocytic cycle within the host, aiding in immune evasion. This rhythmicity, however, diminishes in in vitro cultures, highlighting the importance of host-derived signals for synchronizing the parasite's asexual cycle. Studies indicate a species-specific internal clock in Plasmodium, dependent on these host signals. Melatonin, a hormone the pineal gland produces under circadian regulation, impacts various physiological functions and is extensively reviewed as the primary circadian marker affecting parasite rhythms. Research suggests that melatonin facilitates synchronization through the PLC-IP3 signaling pathway, activating phospholipase C, which triggers intracellular calcium release and gene expression modulation. This evidence strongly supports the role of melatonin as a key circadian marker for parasite synchronization, presenting new possibilities for targeting the melatonin pathway when developing novel therapeutic approaches.

The Role of Neddylation in Malaria Parasites.

Plasmodium parasites, the causative agents of malaria, rely on sophisticated cellular mechanisms to survive and proliferate within their hosts. Plasmodium complex life cycle requires posttranslational modifications (PTMs) to control cellular activities. Neddylation is a type of PTM in which NEDD8 is covalently attached to target proteins and plays an important role in cell cycle control and metabolism. Covalent attachment to its substrates requires the Nedd8-activating enzyme, E1; the NEDD8-conjugating enzyme, E2; and the ligase, E3. In Plasmodium, protein neddylation is essential for parasite development during the stage I-II transition from zygote to ookinete differentiation and malaria transmission. Here, we discuss the current understanding of protein neddylation in Plasmodium, which is involved in malaria transmission.

Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

BACKGROUND: The interaction between iron status and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. METHODS: We retrieved data and samples from 55 participants (19 female) enrolled in malaria VIS, and 171 patients (45 female) with malaria and 30 healthy controls (13 female) enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. FINDINGS: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline ferritin was associated with post-treatment increases in liver transaminase levels. In Malaysian patients with malaria, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. By day 28, hepcidin had normalised; however, ferritin and sTfR both remained elevated. INTERPRETATION: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency. FUNDING: National Health and Medical Research Council (Program Grant 1037304, Project Grants 1045156 and 1156809; Investigator Grants 2016792 to BEB, 2016396 to JCM, 2017436 to MJG); US National Institute of Health (R01-AI116472-03); Malaysian Ministry of Health (BP00500420).

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

Post-Translational Modifications of Proteins of Malaria Parasites during the Life Cycle.

Post-translational modifications (PTMs) are essential for regulating protein functions, influencing various fundamental processes in eukaryotes. These include, but are not limited to, cell signaling, protein trafficking, the epigenetic control of gene expression, and control of the cell cycle, as well as cell proliferation, differentiation, and interactions between cells. In this review, we discuss protein PTMs that play a key role in the malaria parasite biology and its pathogenesis. Phosphorylation, acetylation, methylation, lipidation and lipoxidation, glycosylation, ubiquitination and sumoylation, nitrosylation and glutathionylation, all of which occur in malarial parasites, are reviewed. We provide information regarding the biological significance of these modifications along all phases of the complex life cycle of Plasmodium spp. Importantly, not only the parasite, but also the host and vector protein PTMs are often crucial for parasite growth and development. In addition to metabolic regulations, protein PTMs can result in epitopes that are able to elicit both innate and adaptive immune responses of the host or vector. We discuss some existing and prospective results from antimalarial drug discovery trials that target various PTM-related processes in the parasite or host.

Proteomes of plasmodium knowlesi early and late ring-stage parasites and infected host erythrocytes.

The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

The molecular mechanisms driving Plasmodium cell division.

Malaria, a vector borne disease, is a major global health and socioeconomic problem caused by the apicomplexan protozoan parasite Plasmodium. The parasite alternates between mosquito vector and vertebrate host, with meiosis in the mosquito and proliferative mitotic cell division in both hosts. In the canonical eukaryotic model, cell division is either by open or closed mitosis and karyokinesis is followed by cytokinesis; whereas in Plasmodium closed mitosis is not directly accompanied by concomitant cell division. Key molecular players and regulatory mechanisms of this process have been identified, but the pivotal role of certain protein complexes and the post-translational modifications that modulate their actions are still to be deciphered. Here, we discuss recent evidence for the function of known proteins in Plasmodium cell division and processes that are potential novel targets for therapeutic intervention. We also identify key questions to open new and exciting research to understand divergent Plasmodium cell division.

Epigenetic and transcriptional regulation of cytokine production by Plasmodium falciparum-exposed monocytes.

Plasmodium falciparum infection causes the most severe form of malaria, where excessive production of proinflammatory cytokines can drive the pathogenesis of the disease. Monocytes play key roles in host defense against malaria through cytokine production and phagocytosis; however, they are also implicated in pathogenesis through excessive proinflammatory cytokine production. Understanding the underlying molecular mechanisms that contribute to inflammatory cytokine production in P. falciparum-exposed monocytes is key towards developing better treatments. Here, we provide molecular evidence that histone 3 lysine 4 (H3K4) methylation is key for inflammatory cytokine production in P. falciparum-exposed monocytes. In an established in vitro system that mimics blood stage infection, elevated proinflammatory TNF and IL-6 cytokine production is correlated with increased mono- and tri-methylated H3K4 levels. Significantly, we demonstrate through utilizing a pharmacological inhibitor of H3K4 methylation that TNF and IL-6 expression can be suppressed in P. falciparum-exposed monocytes. This elucidated epigenetic regulatory mechanism, controlling inflammatory cytokine production, potentially provides new therapeutic options for future malaria treatment.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells.

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ T cells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ T cells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites.

IMPORTANCE: In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite-host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host-parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.

The patatin-like phospholipase PfPNPLA2 is involved in the mitochondrial degradation of phosphatidylglycerol during Plasmodium falciparum blood stage development.

Plasmodium falciparum is an Apicomplexa responsible for human malaria, a major disease causing more than ½ million deaths every year, against which there is no fully efficient vaccine. The current rapid emergence of drug resistances emphasizes the need to identify novel drug targets. Increasing evidences show that lipid synthesis and trafficking are essential for parasite survival and pathogenesis, and that these pathways represent potential points of attack. Large amounts of phospholipids are needed for the generation of membrane compartments for newly divided parasites in the host cell. Parasite membrane homeostasis is achieved by an essential combination of parasite de novo lipid synthesis/recycling and massive host lipid scavenging. Latest data suggest that the mobilization and channeling of lipid resources is key for asexual parasite survival within the host red blood cell, but the molecular actors allowing lipid acquisition are poorly characterized. Enzymes remodeling lipids such as phospholipases are likely involved in these mechanisms. P. falciparum possesses an unusually large set of phospholipases, whose functions are largely unknown. Here we focused on the putative patatin-like phospholipase PfPNPLA2, for which we generated an glmS-inducible knockdown line and investigated its role during blood stages malaria. Disruption of the mitochondrial PfPNPLA2 in the asexual blood stages affected mitochondrial morphology and further induced a significant defect in parasite replication and survival, in particular under low host lipid availability. Lipidomic analyses revealed that PfPNPLA2 specifically degrades the parasite membrane lipid phosphatidylglycerol to generate lysobisphosphatidic acid. PfPNPLA2 knockdown further resulted in an increased host lipid scavenging accumulating in the form of storage lipids and free fatty acids. These results suggest that PfPNPLA2 is involved in the recycling of parasite phosphatidylglycerol to sustain optimal intraerythrocytic development when the host resources are scarce. This work strengthens our understanding of the complex lipid homeostasis pathways to acquire lipids and allow asexual parasite survival.

Modulation of Heme-Induced Inflammation Using MicroRNA-Loaded Liposomes: Implications for Hemolytic Disorders Such as Malaria and Sickle Cell Disease.

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMß2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.